B and T lymphocytes are the primary effector cells of the immune responses. Both cell classes are considered to derive ultimately from hematopoietic stem cells in the mammalian bone marrow, via progenitor or precursor cells representing distinguishable stages in the differentiation of each class. Mature T cells develop principally in the thymus, presumably from a precursor cell which migrates from the bone marrow to the thymus at an early stage of T lymphocyte development. Lymphoid cells development is dependent on growth, survival and differentiation factors produced by various stromal cells. Numbers of factors are active on mature peripheral B and T cells, including IL-1, IL-2, IL-4, IL-5, interferon gamma, BSF-2, neuroleukin, transforming growth factor beta and IL-7.
“Interleukin-7” or “IL-7” refers to a mammalian endogenous secretory glycoprotein which is capable of inducing proliferation of bone marrow-derived lymphocyte progenitors and precursors, including the specialized precursors known as pre-B cells. Originally derived from the stromal element of a bone marrow cell line, IL-7 is also secreted by thymic stromal cells, intestinal and other epithelial cells, some dendritic cells and follicular dendritic cells, keratinocytes and generally all lymphoid tissues. Alternative designations for this molecule are “pre-B cell growth factor” and “lymphopoietin-1”.
EP0314415 (or U.S. Pat. No. 4,965,195) describes mammalian interleukin-7 proteins and corresponding DNAs. Human IL-7 amino acid sequence contains three putative N-linked glycosylation sites, located at Asn residues at positions 70, 91 and 116. Transient recombinant expression of hIL-7 (human IL-7) in COS cells allowed the visualization of r-huIL-7 (recombinant human IL-7) as three protein bands of apparent molecular weight of about 20, 24 and 28 kDa (Cosman et al.; Lymphokine Receptor Interactions; 1989; 179:229-236). Stable recombinant expression of hIL-7 in BHK cells was also reported (Armitage et al.; The Journal of Immunology; 1990; 144:938-941). However, the glycosylation status of naturally-occurring human IL-7, particularly the O-glycosylation status, has never been documented or studied, and the impact of the glycosylation profile on IL-7 properties has never been considered. Furthermore, unglycosylated mature human IL-7 (r-hIL-7) produced in E. coli, as described in EP0314415, exhibits a 17,387 Daltons molecular weight and displays a high activity in vitro on specific bioassays based on the proliferation of various lymphocytes populations. Other cytokines and growth factors, such as G-CSF, GM-CSF, IFN, HGF, etc, also display full therapeutic activity without glycosylation.
WO2004/018681 discloses an active conformer of human IL-7, comprising the following disulfide bridges: 1-4 (C2-C92), 2-5 (C34-C129) and 3-6 (C47-C141), as well as methods of producing or characterizing the same and the uses thereof.
IL-7 was originally disclosed as a cytokine whose principal activity was the induction of precursor B cell proliferation (Namen A. E. et al.; Journal of Experimental medicine; 1988; 167:988-1002). IL-7 has more recently been disclosed as being involved in the survival and proliferation of thymocytes (T-Cells) during early stage of T-cell development (Schluns K. S. et al.; Nature Immunology; 2000; 1(5):426-432). IL-7 pathway is essential for lymphocyte development notably on developing thymocytes (Maeurer M. J. et al.; Int. Rev. Immunol.; 1998; 16:309-22-Fry T. J. et al.; Blood; 2002; 99:3892-904). Fry and collaborators further identified IL-7 as a potent modulator of thymic-independent T-cell regeneration in a multifactorial action (Fry T. J. et al.; Blood; 2001; 97(6):1525-1533). IL-7 potently modulates mature T cells and beside this effect on mature T cells, IL-7 may influence the development of antigen presenting cells (Marquez C. et al.; J. Exp. Med.; 1995; 181:475-83). IL-7 is essential for memory T cell regeneration, both in the CD4+ and CD8+ subsets (Kondrack R. M. et al.; J. Exp. Med.; 2003; 198:1797-806-Kaech S. M. et al.; Nat. Immunol.; 2003; 4:1191-8).
IL-7 has thus a great therapeutic potential for use in the stimulation of the proliferation of T cell precursors, of antibody-secreting B cells, in the stimulation of antigen driven T-cell peripheral expansion, and in the production of naïve T-cells as well as other hematopoietic cell types. A particularly interesting therapeutic use of active IL-7 molecules is for immune reconstitution of lymphopenic patients: patients treated for a cancer, patients having received a bone marrow or a stem cell transfer, patients presenting an acquired or genetic immune deficiency, elderly patients or any patients having low CD4 count. Other utilities reside in the ability of IL-7 to produce new naïve CD4 T-cells or to expand specific pools in order to produce or increase specific immune responses (vaccine enhancement).
In view of its therapeutic potentials, there is considerable interest in developing biologically active or improved IL-7 polypeptides that are suitable for efficient therapeutic uses. In this respect, among the various cytokines and growth factors commercially available, some are poorly immunogenic (e.g., Interferon alfa “IFNα”, granulocyte colony stimulating factor “G-CSF”) so that the corresponding drug substances do not require a very specific polypeptide purity other than conventional level usually accepted for recombinant proteins. In contrast, other growth factors are more immunogenic (e.g., Beta interferon “β-IFN”, Granulocyte Macrophage Colony stimulating factor “GM-CSF”) or their specific activity is so critical for life (e.g., Erythropoietin “EPO”) that drug substance polypeptide purity and profile must be specifically studied and maintained inside narrow limits to preserve from immunogenicity.
IL-7 is a unique molecule. Due to its intrinsic immune-enhancing properties, IL-7 used as a therapeutic agent is particularly prone to trigger anti-IL-7 immunogenicity (anti-IL-7 binding or neutralizing antibodies). This immunogenicity is deleterious for the long term therapeutic activity of the protein, Anti-IL-7 antibodies can modify IL-7 pharmacokinetic and neutralize its therapeutic activity.
Various IL-7 isoforms are involved in triggering anti-IL-7 immunogenicity, among which: altered polypeptide sequences (e.g., oxidized, reduced, deamidated or truncated forms), covalent or non covalent IL-7 multimers, such as aggregated IL-7 molecules and the like. Therefore it is critical to define IL-7 polypeptides and drug substances which are more stable, less prone to intermolecular aggregation, less immunogenic and still biologically active. Indeed, while the activity of most drugs is correlated with AUC parameter, the activity of IL-7 is correlated to half-life parameter and more particularly to mean residence time.